Catecholaminergic polymorphic ventricular tachycardia (CPVT; OMIM: 604772) is a highly malignant cardiac disease manifesting in childhood and adolescence. It is characterised by adrenergically-mediated bidirectional or polymorphic ventricular tachycardia leading to syncope and/or sudden cardiac death1, 2. Based on previously reported linkage data that had mapped the disease to chromosome 1q42-433, we reported that the gene for the autosomal dominant variant of CPVT was RyR2; i.e. the gene encoding for the cardiac isoform of the ryanodine receptor4. The first family in which a RyR2 mutation was identified was affected by a highly malignant form of the disease that was resistant to beta-blockers; the mutation present in the family (R4497C) is a hot spot that we subsequently identified in other CPVT patients unrelated to the first kindred. The R4497C mutation has been extensively investigated in different in vitro models that demonstrated that it causes abnormal release of calcium from the sarcoplasmic reticulum5-8. It has been therefore inferred that arrhythmias may develop as a consequence of this defect of intracellular calcium handling. However, experimental evidence linking this mutation to the development of life threatening arrhythmias is still lacking. The cardiac ryanodine receptor (RyR2) is a large tetrameric intracellular calcium (Ca2+) release channel located in the sarcoplasmic reticulum (SR) that has a pivotal role in excitation-contraction coupling. In response to a small intracellular Ca2+ influx through the L-type voltage dependent Ca2+ channels, RyR2 releases from the SR the large amount of Ca2+ that is needed to elicit contraction of the cardiac cell. However, in addition to such a tightly regulated physiological process, RyR2 may also release Ca2+ in response to SR and luminal calcium overload, which may occur under pathological conditions such as physical and emotional stress, digitalis toxicity and heart failure. In such instances RyR2 may become a crucial player for the development of life-threatening arrhythmias.
Previously, we reported that mutations in the gene encoding for RyR2 cause the autosomal dominant form of catecholaminergic polymorphic ventricular tachycardia (CPVT)4. Shortly after, other groups confirmed this finding and reported novel RyR2 mutations in patients affected by CPVT9. More than 20 RyR2 mutations have been reported in the literature10.
The first mutation that we identified in an Italian CPVT family leads to the replacement of arginine at position 4497 with a cysteine. Since this mutation was associated with a very typical CPVT phenotype it has been selected by several authors for their in vitro studies aimed at the functional characterization of RyR2 mutants. Jiang, D. et al., in6 were the first to investigate the R4496C mouse equivalent of the R4497C human mutation. They suggested that when expressed in HEK293 cells the mutation enhances the basal channel activity and the propensity for spontaneous calcium release at rest and in response to caffeine. More recently, the same authors further elaborated their results and proposed that the R44967C (or its murine homolog R4496C), as well as other RyR2 mutations identified in CPVT families, increase the sensitivity of RyR2 channels to luminal [Ca2+] thus facilitating the spontaneous release of Ca2+ from the SR5. George et al7 investigated the same mutation by expression in HL-1 cardiac myocytes. At variance with what was suggested by Jiang et at5 based on their studies in HEK 293 cells, George et al.7 reported that the R4496C mutant presents no enhancement of basal activity but they confirmed that after exposure to the RyR agonist caffeine or to beta adrenergic stimulation, calcium release was significantly augmented in the mutant channels. George et al also showed that the dissociation of the FKBP12.6 protein from the mutant was similar to that observed in the WT RyR2 thus challenging the hypothesis advanced by Wehrens et al8 who proposed that the enhanced calcium release observed in the mutant during beta adrenergic stimulation was caused by the excessive dissociation of the RyR2:FKBP12.6 complex. Overall, although disagreement exists on the mechanisms by which the R4496C mutation sensitizes the RyR2 channel to agonists, three independent groups have confirmed that upon caffeine and beta adrenergic stimulation RyR2R4496C channels respond with an augmented calcium release.
The large amount of data reported in the last few years suggest that RyR2-R4497C (or its murine homolog R4496C) is a kind of prototype among RyR2 mutation very suitable to study the mechanisms cardiac arrhythmias due to intracellular Ca2+ handling abnormalities.
Here we report on a conditional knock-in mouse model carrier of the R4496C mutation that is the mouse equivalent of the human mutation R4497C. The aim of the present work is to generate an animal model of CPVT allowing to better characterize the clinical phenotype, the pathogenetic mechanisms and to gather insights on possible novel therapies of CPVT and RyR2-mediated arrhythmogenesis in general.